A CURE for Microbiology Experiment: Isolation and Characterization of Antimicrobial‐Producing Lactic Acid Bacteria From Fermented Sichuan Pickle Against Pathogenic Bacteria

ABSTRACT

Traditional microbiology experimental teaching methodology, often based on rigid “cookbook” experiment plans, fails to meet the growing need to cultivate skilled and innovative individuals. Course‐based undergraduate research experiences (CUREs) provide a solution by integrating research‐based learning into the curriculum, offering an opportunity to improve the “hard” and “soft” skills for science, technology, engineering, and mathematics (STEM) students. In this study, a CURE was incorporated into the microbiology experiment curriculum focusing on the isolation and characterization of antimicrobial‐producing lactic acid bacteria (LAB) from fermented Sichuan pickles. The CURE was divided into four key sections: (i) designing an experimental plan, (ii) isolation of LAB, (iii) evaluating the antibacterial effects and antibacterial active substances of LAB, and (iv) identifying LAB species and completing the experimental report. Through a tutor observation of the experiment process and a student questionnaire survey, the study demonstrated that CURE‐based microbiology experiments significantly improved students' laboratory skills and microbiological‐related experimental operation ability and nurtured their scientific thinking. This approach enhances students' preparedness for future career success and cultivates the growth of a well‐rounded, skilled person in STEM disciplines.

1. Introduction

Conventional microbiology education plays a pivotal role in various fields, including healthcare, industrial manufacturing, agricultural production, environmental protection, and fundamental science research. Its advancement promotes progress in human society and technology. Microbiology experiments serve as a crucial link between microbiological theory and practice, contributing significantly to the field's development [1]. Therefore, it is crucial to cultivate skilled individuals proficient in microbiology techniques to address microbial‐related challenges and promote progress in microbiology. Universities and colleges form the foundation for talent development and serve as the primary institutions for nurturing individuals equipped to meet societal needs [2]. Traditionally, microbiology experimental courses in higher education institutions such as universities and colleges follow predesigned protocols with outcomes known to instructors [3, 4]. This approach fails to effectively engage students' interest in learning and does not sufficiently foster the development of their experimental skills or scientific reasoning. In addition, microbiology courses often consist of independent experiments that lack cohesion and systematic training, resulting in students' inability to apply taught concepts in practice. To address these shortcomings, it is essential to adopt new teaching methodologies to improve the effectiveness of microbiology experiment instruction.

Various advanced teaching methodologies have been developed and implemented to improve the teaching efficacy of microbiology laboratory courses. For instance, Zhang et al. [5] implemented an innovative online‐to‐offline teaching approach in a veterinary microbiology course, which enhanced students' learning performance and professional recognition by incorporating massive open online courses (MOOCs) as preparatory material prior to in‐person teaching. A video‐based teaching strategy was also developed to increase efficiency in microbiology experiments [6]. In addition, a teaching model integrating virtual simulations with real experiments was implemented in a clinical microbiology course to improve students' operational skills and theoretical knowledge [7]. Course‐based undergraduate research experiences (CUREs) have gained significant attention in higher education due to their mutual benefit for both students and tutors, as well as their proven effectiveness [8, 9, 10, 11]. CUREs offer an inquiry‐based learning approach that provides students with research experience during their undergraduate studies [1, 12]. In a CURE, students are motivated by scientific questions to engage in studies where the answers are often unknown to both the students and the tutors [13, 14]. CUREs provide students with practical experience in formulating research questions, designing experiments, analyzing data, and addressing scientific questions [15, 16, 17, 18], making them especially suitable for laboratory‐learning courses [19]. D'Angelo et al. [16] demonstrated that a CURE‐based traditional organic chemistry laboratory enhanced student engagement and learning outcomes. Similarly, a CURE‐based microbial engineering course improved students' understanding of fermentation processes, developed their engineering skills, and cultivated research skills and scientific attitudes [1]. A project focused on identifying vaginal microorganism species communities implemented CURE methodology to enhance students' scientific literacy skills [20]. To date, most successful CUREs have been implemented in chemistry, environmental science, and clinical microbiology. However, the impact of CUREs varies across different disciplines and contexts [21]. CURE modules tailored to food fermentation systems remain scarce, particularly those relevant to biochemistry and molecular biology knowledge systems.

Lactic acid bacteria (LAB) are important functional microorganisms widely distributed in fermented foods. Many LAB produce antimicrobial substances and show strong inhibitory activity against foodborne pathogens, making them valuable for food preservation and safety [22, 23, 24]. Traditional Chinese fermented foods represented by Sichuan pickle are rich in LAB resources and highly suitable for undergraduate experimental teaching. However, almost no CURE modules have been established to use Sichuan pickles as a teaching model to systematically guide undergraduates in completing the whole process of LAB isolation, antibacterial activity detection, antibacterial component analysis, and molecular identification.

This study proposes a CURE‐based microbiology experiment focused on the isolation and characterization of LAB with antibacterial properties. The learning objectives of this CURE module are to stimulate students' interest in microbiological experimentation, strengthen their practical laboratory skills, and cultivate scientific literacy. More specifically, students will master a series of core experimental skills covering microbiology and biochemistry. These techniques include strain isolation, aseptic manipulation, Gram staining, optical microscopy, the Oxford cup antibacterial assay, and minimum inhibitory concentration (MIC)/minimum bactericidal concentration (MBC) determination. Students will also understand the biochemical basis of LAB antimicrobial metabolites and grasp the molecular principles of 16S rRNA‐based bacterial identification. Furthermore, students can effectively improve their comprehensive soft skills, including academic communication, result presentation, and collaborative teamwork. To achieve these learning objectives, students were primarily responsible for designing the experiment plan, conducting the procedures, writing the experiment reports, and providing evaluations, while tutors guided the research direction, revised experimental procedures, and supervised the experimental process. This course provided students without prior research experiences the opportunity to participate in research and develop the ability to integrate theory with practical situations.

2. Methods

2.1. Course Overview

Microbiology experiment is a compulsory course taught to second‐year food science and engineering students. This experiment can be completed in approximately 24 h and is divided into four units. The whole microbiology experiment consists of four sessions: (i) designing the experiment plan, (ii) isolation and preliminary identification of antimicrobial‐producing LAB, (iii) evaluation of antimicrobial performance and antibacterial active substances of LAB, and (iv) identification of LAB species and completion of the experiment report. Table 1 presents the detailed timeline and grouping arrangement of the CURE experiment. The students worked in groups of six throughout the experiment. To improve the efficiency of the experiment, specific arrangements were made in the experiment process. First, all the students in each group collaborated to complete the first session: designing the experiment plan (one unit took 6 h). In the second session, each group was divided into three subgroups to handle isolation and preliminary identification of microbiology. Two students were responsible for isolation, two for preliminary identification, and the remaining two for the Oxford cup experiment (one unit, 6 h). For the third session (one unit, 8 h), the group was divided into two subgroups: three students focused on determining the MIC and MBC, while the other three students investigated the antibacterial substances in the fermentation supernatant. These experiments were performed simultaneously to ensure all experiments were completed within the stipulated time frame. Finally, LAB strains with potent antibacterial activity were selected for species identification, followed by the completion of the experimental report (one unit, 4 h).

TABLE 1.

Timeline and grouping arrangement of the CURE experiment.

Units	Activities	Group arrangement
1 (6 h)	Designing the experiment plan	All 6 students worked together to develop the experimental scheme
2 (6 h)	Isolation and preliminary identification of antimicrobial‐producing LAB	Divided into three 2‐student subgroups: one for strain isolation, one for preliminary identification, and one for the Oxford cup assay
3 (8 h)	Evaluation of antimicrobial performance and antibacterial active substances of LAB	Divided into two 3‐student subgroups: one for MIC/MBC determination, and the other for investigating antibacterial substances in fermentation supernatants
4 (4 h)	Identification of LAB species and completing the experiment report	All students selected strains for species identification and completed individual experimental reports

2.2. Course Design

The first session focused on guiding students in designing and refining their experimental plans. First, 10 research papers, closely related to the experiments, were provided as references to assist students in developing their experimental plan. Students were then required to create a PowerPoint presentation outlining their experimental plan, with one student responsible for explaining the purpose, principles, methods, materials, and steps involved in the experiment. During the presentation, other students posed questions about the experimental plan, followed by a discussion with instructors to ensure a deeper understanding of the experiment's principles and the importance of each step.

The second session involved the isolation of antimicrobial‐producing LAB, as described in Data S1. In alignment with the tutors' research projects, LAB strains with antibacterial properties were expected to be isolated from naturally fermented Sichuan pickles. Students collected the naturally fermented Sichuan pickles using sterile bags under aseptic conditions. MRS medium containing 1.5% CaCO₃ was prepared and used for the initial isolation of LAB. The Gram‐staining method was employed to examine the colony morphology of the isolated strains. Simultaneously, the Oxford cup method was used to evaluate the antibacterial effect of the isolated LAB against Staphylococcus aureus and Escherichia coli O157:H7.

The third session focused on evaluating the antibacterial properties of the LAB fermentation supernatant, including determining the MIC and MBC, as well as investigating the antibacterial active substances present in the supernatant. A classical 96‐well plate‐based method, detailed in Data S1, was used to measure the MIC and MBC. Common antibacterial active substances in LAB fermentation supernatants include organic acids, hydrogen peroxide, and bacteriocins [25]. To assess their roles, exclusion experiments were conducted to investigate the contributions of these substances to the antibacterial activity.

The final session involved identifying the species of LAB and completing the experimental report. Following the evaluation of antibacterial properties, 16S rRNA gene sequencing was employed for species identification of LAB. A single LAB colony was selected and cultured in MRS medium at 37°C overnight. Finally, the fresh cultured LAB was sequenced by Chengdu Tsingke BioTech Co. (Chengdu, China).

All quantitative laboratory experiments in this study, including antibacterial activity assays, MIC/MBC determinations, and investigation of antibacterial substances in fermentation supernatants, were performed in three independent biological replicates with strict negative control groups. Routine qualitative procedures such as strain isolation, purification, and Gram staining were conducted following standard microbiological protocols, which do not require redundant biological replicates, as they yield stable and consistent morphological results. This standardized arrangement ensures the reliability and reproducibility of quantitative data and also helps students establish rigorous scientific thinking, standard experimental norms, and a clear understanding of when to apply experimental repetition and control groups in research.

2.3. Hazards

During the experiments, students were required to wear laboratory coats and gloves at all times. These items must be left in the laboratory upon leaving. Students should exercise caution when using the automatic autoclave, as it produces steam during the sterilization process. Proper handling of alcohol lamps is essential to avoid burns, and ethanol, being highly flammable, must be kept away from open flames. The bacterial strains E. coli O157:H7 CMCC44102 and S. aureus ATCC 29213 are classified as biosafety level (BSL)‐2 organisms. Therefore, all experiments involving these bacteria were conducted in a Class II biosafety cabinet, which was sterilized after each experiment.

3. Results and Discussion

3.1. Designing Experimental Plans

Before conducting the experiments, students were required to develop a detailed experimental plan based on their review of scientific literature. This process helped students understand the importance of scientific literature when entering an unfamiliar area of scientific research. The experimental plans were then presented in a PowerPoint presentation, with one student explaining the purpose, principles, methods, materials, and steps involved. During the presentations, other students had an opportunity to ask questions, which were discussed with the tutors, deepening the students' understanding of the experimental procedure and scientific research principles. These activities provided students with an opportunity to engage in scientific research, fostering their scientific thinking and teamwork abilities.

3.2. Isolation of LAB With Antimicrobial Activity

Sichuan pickles are a popular traditional fermented food in China, made through natural fermentation, which contains various LAB. Using naturally fermented Sichuan pickles as the experimental object would increase the likelihood of experimental success. The pickle samples were collected by students from their families under aseptic conditions. This emphasized the importance of sterile operation in microbiology experiments, given the potential presence of environmental microorganisms. The students prepared MRS medium with 1.5% CaCO₃ to isolate LAB. Students mastered the procedures for medium preparation, autoclave operation, and strain isolation and developed a strong awareness of aseptic operation throughout the process. In one group, six bacteria strains were selected based on the size of their soluble calcium circle for subsequent preliminary identification (Figure 1a). The colonies exhibited a similar round, milky white, moist, and smooth surface with neat edges. Gram staining was then used for preliminary identification, and all strains were found to be Gram‐positive bacteria, rod‐shaped structures, similar to the morphology of Bacterial Strain 1 (Figure 1b). Students learned how to perform Gram staining and use an optical microscope since Gram staining is a very important and basic method for bacterial identification in microbiology experiments. Variations in bacterial size viewed under the optical microscope were different, confirming that different LAB strains were present (data not shown).

FIGURE 1.

The soluble calcium circle of bacterial strains (A), Gram‐staining of Bacterial Strain 1 (B).

Next, the antimicrobial activity of LAB fermentation supernatants was evaluated against E. coli O157:H7 (Gram‐negative bacteria) and S. aureus (Gram‐positive bacteria) using the Oxford cup method. Students were instructed on key experimental steps, including preparing the LAB fermentation supernatant, determining bacterial concentrations, and spreading E. coli O157:H7 and S. aureus evenly on the agar plates. In this process, students learned standard operating procedures for the Oxford cup method, mastered the evaluation of antimicrobial activity, and developed a good awareness of aseptic manipulation. The obtained results were expressed as the growth inhibition diameter of each fermentation supernatant against E. coli O157:H7 or S. aureus (mm) (Figure 2). The students compared that LAB fermentation supernatant inhibited E. coli O157:H7 and S. aureus while the MRS medium (negative control) had no effect, as indicated by an inhibition zone diameter of 8 mm (the diameter of the Oxford cup) (Table 2). All the groups performed this experiment and obtained similar results demonstrating that LAB produced antibacterial substances, though the antibacterial efficacy varied among strains. For example, LAB Strain 5 against E. coli O157:H7 and S. aureus showed an inhibition zone diameter of 15–20 mm, while the two strains LAB Strains 2 and 4 had an inhibition zone diameter of 20–25 mm. Notably, Strains 1 and 3 exhibited the largest inhibition zones, exceeding 25 mm, indicating strong antibacterial ability against E. coli O157:H7 and S. aureus . Specifically, the inhibition zone diameters of LAB Strain 2 against pathogenic bacteria ranged from 23 to 27 mm, while Strains 1 and 3 both produced larger inhibition zone diameters (> 25 mm) against E. coli O157:H7 and S. aureus .

FIGURE 2.

The inhibition zone of Strain 1 fermentation supernatant against Escherichia coli (A) and Staphylococcus aureus (B). The top left corner of each plate was MRS (negative control); the rest are three parallel samples of fermentation supernatant.

TABLE 2.

Antibacterial activity of LAB fermentation supernatant against Escherichia coli O157:H7 and Staphylococcus aureus .

LAB strain no.	The diameter of inhibitory zone (mm)
	E. coli O157:H7	S. aureus
Strain 1	26.37 ± 0.78	26.13 ± 0.91
Strain 2	23.20 ± 0.56	26.17 ± 0.72
Strain 3	26.23 ± 0.68	25.77 ± 0.84
Strain 4	22.97 ± 0.84	23.10 ± 1.65
Strain 5	17.70 ± 1.06	19.57 ± 0.74
Strain 6	21.40 ± 0.66	20.07 ± 0.95
Negative control	8.03 ± 0.83	8.07 ± 0.32

3.3. Evaluation of the Antibacterial Properties and Antibacterial Active Substances of LAB

To further compare the antibacterial activities, the MIC and MBC of the LAB fermentation supernatant were measured. The students learned to standardize LAB fermentation concentrations and observe turbidity in the 96‐well plates under different concentrations to determine the MIC. The lowest concentration at which the solution remained clear and transparent was recorded as the MIC of LAB fermentation for pathogenic bacteria (Figure 3A). Meanwhile, students also learned the experimental principle and operational criteria for determining MBC through plate coating and colony observation. As shown in Figure 3B, after 24 h of incubation at 37°C, the corresponding lowest concentration at which no bacterial colonies formed was observed on an LB agar, which was the MBC of LAB fermentation supernatant for pathogenic bacteria. As shown in Table 3, the MICs of the fermentation supernatant from Strains 1–6 against E. coli were 8.00 mg/mL for Strains 1–4 and 128.00 mg/mL for Strains 5–6, respectively. The MBCs increased to 8.00 mg/mL for Strain 1, 16.00 mg/mL for Strains 2–4, and 256.00 mg/mL for Strains 5–6. For S. aureus , the MICs were 8.00 mg/mL for Strains 1 and 3, 16.00 mg/mL for Strains 2 and 4, and 128.00 mg/mL for Strains 5 and 6, while the MBCs were 16.00 mg/mL for Strains 1 and 3, 32.00 mg/mL for Strains 2 and 4, and 256.00 mg/mL for Strains 5 and 6. Among the fermentation supernatant Strains 1–6, Strain 1 demonstrated the strongest antibacterial activity against both E. coli O157:H7 and S. aureus .

FIGURE 3.

The MIC and MBC of fermentation supernatant of Strain 1 against Escherichia coli.

TABLE 3.

The MIC and MBC of LAB fermentation supernatant against Escherichia coli and Staphylococcus aureus .

LAB strain no.	MIC (mg/mL)		MBC (mg/mL)
	E. coli O157:H7	S. aureus	E. coli O157:H7	S. aureus
Strain 1	8.00	8.00	8.00	16.00
Strain 2	8.00	16.00	16.00	32.00
Strain 3	8.00	8.00	16.00	16.00
Strain 4	8.00	16.00	16.00	32.00
Strain 5	128.00	128.00	256.00	256.00
Strain 6	128.00	128.00	256.00	256.00

To investigate the antibacterial active substances in the fermentation supernatants of six LAB strains, the supernatants were treated with a base, catalase, and protease (trypsin and pepsin), respectively. This experimental design allowed students to apply scientific thinking and methodologies to achieve the desired results. As shown in Table 4, the inhibition zone diameter of six LAB fermentation supernatants against E. coli decreased after the fermentation supernatant was treated with the base, catalase, and protease (pepsin and trypsin). This reduction indicates that the antibacterial active substances present were likely organic acid, hydrogen peroxide, and bacteriocins. Furthermore, after successive treatments with the base, catalase, and protease (pepsin and trypsin), the inhibition zone diameter of six LAB fermentation supernatants was almost the same as the diameter of the Oxford cup, suggesting a complete loss of the antibacterial ability of fermentation supernatants. Thus, the results confirm that the antibacterial active substances in fermentation supernatants are limited to three components: acid, hydrogen peroxide, and bacteriocins.

TABLE 4.

The inhibition zone diameter of six LAB fermentation supernatants against Escherichia coli after fermentation supernatant was treated under different conditions.

Treatment condition	The diameter of inhibition zone (mm)
	Strain 1	Strain 2	Strain 3	Strain 4	Strain 5	Strain 6
Without treatment	25.40 ± 0.78	23.43 ± 0.38	24.50 ± 1.04	25.23 ± 0.21	18.73 ± 0.38	18.13 ± 0.40
Base	15.73 ± 0.49	17.03 ± 0.87	17.46 ± 0.75	16.70 ± 0.96	17.87 ± 0.51	18.33 ± 0.65
Catalase	21.90 ± 0.21	20.73 ± 0.25	20.53 ± 0.49	19.96 ± 0.45	16.97 ± 0.42	17.17 ± 0.85
Trypsin	20.57 ± 0.45	19.60 ± 0.36	19.93 ± 0.38	20.13 ± 0.32	15.90 ± 0.80	15.12 ± 0.57
Pepsin	20.33 ± 0.32	19.10 ± 0.36	19.37 ± 0.72	19.40 ± 0.66	15.36 ± 0.77	15.87 ± 0.35
Combined treatment	8.10 ± 0.45	8.09 ± 0.36	8.13 ± 0.56	8.08 ± 0.78	8.01 ± 0.76	8.05 ± 0.56

3.4. Identification of LAB Species

Finally, 16S rRNA gene sequencing was used to identify the species of strains with strong antibacterial ability. Students were taught the principles of bacterial identification using 16S rRNA gene sequencing, and the obtained sequences were aligned using BLAST software at NCBI GenBank, and the phylogenetic tree of bacteria was constructed using MEGA7 software. Students learned how to use both software tools, BLAST and MEGA7. As shown in Figure 4, Strain 1 shared 99% similarity with Lactobacillus plantarum strain JCM 1149.

FIGURE 4.

The phylogenetic tree of Strain 1 based on 16S rDNA sequence.

3.5. Feedback of Students About CURE‐Based Microbiology Experiment

Finally, a questionnaire survey was given to 30 students to assess their impressions and learning outcomes of the experiments. As shown in Table 5, students reported that they had significantly improved their microbiological‐related experimental skills, or “hard skills,” and found the experiment very interesting. Although some students considered the experiment challenging, it helped develop their communication ability, expression abilities, and critical scientific thinking abilities, thereby improving their “soft skills.” For most students, this was their first experience participating in scientific research, which broadened their horizons and sparked their interest in pursuing further scientific research.

TABLE 5.

The students' impression and learning outcome of the experiments.

Questions	1 indicates	5 indicates	Mean score (N = 30)
Q1: How interested you are in the experiment?	Not at all	Very interesting	4.63 ± 0.56
Q2: Were you familiar with the experiment plan?	Not at all	Very familiar	4.93 ± 0.41
Q3: Were you familiar with the basic operation of microbiological experiment? Such as medium preparation, bacterial inoculation and culture, streak plate methods, spread plate method, Gram staining	Not at all	Very familiar	4.95 ± 0.23
Q4: Were you familiar with oxford cup method?	Not at all	Very familiar	4.91 ± 0.78
Q5: Do you know how to obtain the MIC and MBC of antibacterial substance?	No	Yes	4.89 ± 0.11
Q6: Do you think teamwork is helpful?	No	Yes	4.83 ± 0.45
Q7: Do you think this teaching methodology is useful to your undergraduate study?	Disagree	Agree	4.82 ± 0.32

4. Conclusion

A systematic and comprehensive experiment was designed for second‐year undergraduate students, including isolation of LAB, evaluation of their performance, and subsequent identification of LAB species, which benefited both students and tutors. The tutors successfully completed the course, isolating several high‐performance LAB strains that can be used for further research. For the students, this experiment improved their practical skills, communication and presentation abilities, and scientific literacy skills that will contribute to their future career success and development as competent professionals.

Funding

This work was supported by Xihua University (xjjg2025100).

Ethics Statement

This study was a routine curriculum‐based teaching practice involving student participation. All activities did not collect any identifiable personal information of students, involved minimal risk, and did not belong to human biomedical research. Therefore, this project was granted an exemption from ethical review by the Institutional Ethics Committee of Xihua University.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1: Supporting Information.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.